1. Field of the Invention
The present invention relates to a cell in which the activity of a protein relating to transport of an intracellular sugar nucleotide, GDP-fucose, to the Golgi body is more decreased or deleted than its parent cell; a process for producing an antibody composition using the cell; a transgenic non-human animal or plant or the progenies thereof, in which genome is modified so as to have a decreased or deleted activity of a protein relating to transport of an intracellular sugar nucleotide, GDP-fucose, to the Golgi body; a process for producing an antibody composition from the animal or plant; and a medicament comprising the antibody composition.
2. Brief Description of the Background Art
In the Fc region of an antibody of an IgG type, two N-glycoside-linked sugar chain binding sites are present. In serum IgG, to the sugar chain binding site, generally, binds a complex type sugar chain having plural branches and in which addition of sialic acid or bisecting N-acetylglucosamine is low. It is known that there is variety regarding the addition of galactose to the non-reducing end of the complex type sugar chain and the addition of fucose to the N-acetylglucosamine in the reducing end [Biochemistry, 36, 130 (1997)].
It has been considered that such a structure of a sugar chain is determined by a glycosyltransferase which synthesizes a sugar chain and a glycolytic enzyme which hydrolyzes the sugar chain.
Synthesis of an N-glycoside-linked sugar chain is described below.
Glycoproteins are modified with a sugar chain in the endoplasmic reticulum (hereinafter referred to as “ER”) lumen. During the biosynthesis step of the N-glycoside-linked sugar chain, a relatively large sugar chain is transferred to the polypeptide chain which is elongating in the ER lumen. In the transformation, the sugar chain is firstly added in succession to phosphate groups of a long chain lipid carrier comprising about 20 α-isoprene units, which is called dolichol phosphate (hereinafter sometimes referred to as “P-Dol”). That is, N-acetylglucosamine is transferred to dolichol phosphate to thereby form GlcNAc-P-P-Dol and then one more GlcNAc is transferred to form GlcNAc-GlcNAc-P-P-Dol. Next, five mannoses (hereinafter mannose is also referred to as “Man”) are transferred to thereby form (Man)5-(GlcNAc)2-P-P-Dol and then four Man's and three glucoses (hereinafter glucose is also referred to as “Glc”) are transferred. Thus, a sugar chain precursor, (Glc)3-(Man)9-(GlcNAc)2-P-P-Dol, called core oligosaccharide is formed. The sugar chain precursor comprising 14 sugars is transferred as a mass to a polypeptide having an asparagine-X-serine or asparagine-X-threonine sequence in the ER lumen. In the reaction, dolichol pyrophosphate (P-P-Dol) bound to the core oligosaccharide is released but again becomes dolichol phosphate by hydrolysis with pyrophosphatase and is recycled. Trimming of the sugar chain immediately starts after the sugar chain binds to the polypeptide. That is, 3 Glc's and 1 or 2 Man's are eliminated on the ER, and it is known that α-1,2-glucosidase I, α-1,3-glucosidase II and α-1,2-mannosidase relates to the elimination.
The glycoprotein which was subjected to trimming on the ER is transferred to the Golgi body and are variously modified. In the cis part of the Golgi body, N-acetylglucosamine phosphotransferase which relates to addition of mannose phosphate, N-acetylglucosamine 1-phosphodiester α-N-acetylglucosaminidase and α-mannosidase I are present and reduce the Man residues to 5. In the medium part of the Golgi body, N-acetylglucosamine transferase I (GnTI) which relates to addition of the first outside GlcNAc of the complex type N-glycoside-linked sugar chain, α-mannosidase II which relates to elimination of 2 Man's, N-acetylglucosamine transferase II (GnTII) which relates to addition of the second GlcNAc from the outside and α1,6-fucosyltransferase which relates to addition of fucose to the reducing end N-acetylglucosamine are present. In the trans part of the Golgi body, galactose transferase which relates to addition of galactose and sialyltransferase which relates to addition of sialic acid such as N-acetylneuraminic acid are present. It is known that N-glycoside-linked sugar chain is formed by activities of these various enzymes.
Regarding the sugar chain of an antibody, Boyd et al. have examined effects of a sugar chain on the antibody-dependent cell-mediated cytotoxic activity (hereinafter referred to as “ADCC activity”) and complement-dependent cytotoxic activity (hereinafter referred to as “CDC activity”) by treating a human CDR-grafted antibody CAMPATH-1H (human IgG1 subclass) produced by a Chinese hamster ovary cell (CHO cell) or a mouse myeloma produced by NS0 cell with various sugar hydrolyzing enzymes, and reported that elimination of the non-reducing end sialic acid did not have influence upon both activities, but the CDC activity alone was affected by further removal of galactose residue and about 50% of the activity was decreased, and that complete removal of the sugar chain caused disappearance of both activities [Molecular Immol., 32, 1311 (1995)]. Also, Lifely et al. have analyzed the sugar chain bound to a human CDR-grafted antibody CAMPATH-1H (human IgG1 subclass) which was produced by CHO cell, NS0 cell or rat myeloma YO cell, measured its ADCC activity, and reported that the CAMPATH-1H produced by YO cell showed the highest ADCC activity, suggesting that N-acetylglucosamine (hereinafter sometimes referred to as “GlcNAc”) at the bisecting position is important for the activity [Glycobiology, 5, 813 (1995); WO 99/54342].
Furthermore, regarding a sugar chain in an antibody, it is reported that addition-modification of fucose to N-acetylglucosamine in the reducing end in the N-glycoside-linked sugar chain of an antibody changes the ADCC activity of the antibody greatly (WO00/61739). These reports indicate that the structure of the sugar chain plays an important role in the effector functions of human antibodies of IgG1 subclass.
In general, most of the humanized antibodies of which application to medicaments is in consideration are prepared by using genetic recombination techniques and produced by using Chinese hamster ovary tissue-derived CHO cell as the host cell. However, as described above, since the sugar chain structure plays a remarkably important role in the effector function of antibodies and differences of the sugar chain structure of glycoproteins depend on host cells which produce the glycoproteins, development of a host cell which can be used for the production of an antibody having higher effector function is desired.
In order to adjust the activity of an enzyme relating to modification of a sugar chain in a host cell and modify the sugar chain structure of the produced glycoprotein, a method in which an inhibitor against an enzyme relating to the modification of a sugar chain is applied has been attempted.
Examples of an inhibitor against an enzyme relating to the modification of a sugar chain include tunicamycin which selectively inhibits formation of GlcNAc-P-P-Dol which is the first step of the formation of a core oligosaccharide which is a precursor of an N-glycoside-linked sugar chain, castanospermin and N-methyl-1-deoxynojirimycin which are inhibitors of glycosidase I, bromocondulitol which is an inhibitor of glycosidase II, 1-deoxynojirimycin and 1,4-dioxy-1,4-imino-D-mannitol which are inhibitors of mannosidase I, swainsonine which is an inhibitor of mannosidase II and the like. Examples of an inhibitor specific for a glycosyltransferase include deoxy derivatives of substrates against N-acetylglucosamine transferase V (GnTV) and the like [Glycobiology Series 2-Destiny of Sugar Chain in Cell (Kodan-sha Scientific), edited by Katsutaka Nagai, Senichiro Hakomori and Akira Kobata (1993)]. Also, it is known that 1-deoxynojirimycin inhibits synthesis of a complex type sugar chain and increases the ratio of high mannose type and hybrid type sugar chains. Actually, it has been reported that sugar chain structure of IgG produced by a hybridoma was changed and properties such as antigen binding activity or DCC activity were changed when the inhibitors such as castonospermine, N-methyl-1-deoxynojirimycin, swainsonine and tunicamycin were added to a medium [Molecular Immunol., 26, 1113 (1989)]. However, since these inhibitors have weak specificity and also cannot inhibit the target enzyme sufficiently, it is difficult to surely control the sugar chain structure of the produced antibody.
Also, an attempt has been made to modify the sugar chain structure of a produced glycoprotein by introducing an enzyme gene relating to the modification of sugar chains into the host cell, and specifically, it has been reported that 1) it is possible to produce a protein in which sialic acid is added in a large number to the non-reducing end of a sugar chain by introducing rat β-galactoside-α-2,6-sialyltransferase into CHO cell [J. Biol. Chem., 261, 13848 (1989)], 2) it is possible to express an H antigen in which fucose (hereinafter also referred to as “Fuc”) is added to the non-reducing end of a sugar chain (Fucα1-2Galβ1-) by introducing human β-galactoside-2-α-fucosyltransferase into mouse L cell [Science, 252, 1668 (1991)], and 3) it is possible to produce an antibody having a high addition ratio of the bisecting N-acetylglucosamine of N-glycoside binding sugar chains by producing an antibody using a β-1,4-N-acetylglucosamine transferase III (GnTIII)-introduced CHO cell [Glycobiology, 5, 813 (1995): WO 99/54342]. When the antibody was expressed by using a GnTIII-introduced CHO cell, it showed 16 times higher ADCC activity than the antibody expressed in the parent cell. However, since it has been reported that over-expression of GnTIII or β-1,4-N-acetylglucosamine transferase V (GnTV) shows toxicity upon CHO cell, it is not suitable for the production of antibody medicaments.
It has also been reported on a production example of a glycoprotein in which a produced sugar chain structure was changed by using, as a host cell, a mutant in which the activity of an enzyme gene relating to the modification of sugar chains was changed, and as its example, it has been reported that an antibody having a high mannose type sugar chain structure was produced by using a mutant clone of CHO cell in which the activity of 4-N-acetylglucosamine transferase I (GnTI) [J. Immunol., 160, 3393 (1998)] was deleted. In addition, expression of an antibody having a sugar chain structure in which sialic acid is not bound to the non-reducing side in the sugar chain and an expression example of an antibody having a sugar chain structure to which galactose is not bound, by using a CMP-sialic acid transporter- or UDP-galactose transporter-deficient cell line, respectively, have been reported, but no antibody having improved effector functions suitable for the application to medicaments has been found [J. Immunol., 160, 3393 (1998)]. Since the mutant clones have been obtained as clones resulting from the introduction of random mutation by mutagen treatment, they are not suitable as clones used in the production of pharmaceutical preparations.
Thus, in order to modify a sugar chain structure of a produced glycoprotein, attempts have been made to control the activity of an enzyme relating to the modification of sugar chains in host cells. However, in fact, since the sugar chain modification mechanism is varied and complicated and it cannot be said that physiological roles of sugar chains has been sufficiently revealed, it is the present situation that trial and error are repeated. Particularly, it has been revealed gradually that effector functions of antibodies have great influences by sugar chain structures, but a host cell capable of producing antibody molecules modified with a most suitable sugar chain structure has not been obtained yet.